Tumor-induced immune evasion represents one chief barrier in cancer immunotherapy. The mechanisms underlying this immune evasion include defective tumor-antigen processing and presentation, production of immune inhibitory cytokines, and induction of tumor-specific T cell tolerance mediated by CD4+CD25+ regulatory T (TReg) cells. Myeloablative chemotherapy with stem cell transplant may offer the best chance of achieving a state of minimal residual disease, and thus minimizing tumor-induced immune evasion. However, TReg-mediated tumor-specific T cell tolerance persists following transplant. We have identified that in vivo stimulation of Toll-like receptors (TLRs) of the innate immunity is required for the reversal of TReg-mediated suppression on tumor-specific CD8+ T cells post-transplant. This can be achieved by conventional dendritic cell (cDC) vaccines co-administered with a TLR9 ligand, CpG in vivo. We have also discovered that vaccinia virus (VV)-based vaccines can even more potently activate tumor-specific CD8 T cell response post-transplant due to their unique ability to activate multiple innate immune pathways in vivo: VV activates cDCs through TLR2, leading to the production of pro-inflammatory cytokines, and a TLR-independent pathway, resulting in secretion of type I interferons (IFNs); In addition, VV also activates plasmacytoid DCs (pDCs) to secrete high levels of type I IFNs in a TLR-dependent manner. Taken together, these observations suggest a central hypothesis that efficient activation of innate immune system is critical for overcoming TReg-mediated suppression on tumor- specific T cells through post-transplant vaccinations. To test this hypothesis, we propose to pursue the following 5 aims: 1) To study the biological function of pDCs in TLR-dependent augmentation of anti-tumor immunity post-transplant; 2) To delineate the mechanism(s) underlying TLR-dependent reversal of TReg-mediated suppression on tumor-specific T cells post-transplant; 3) To investigate the synergistic effect of multiple TLRs on the generation of anti-tumor effector and long-lived memory T cells post-transplant; 4) To determine the function of TLRs in the recovery of innate immune cells post-transplant; 5) To evaluate the efficacy of novel immunotherapeutic strategies for treating pre-established tumors in the setting of stem cell transplant. We expect that this work will lead to the identification of critical elements for enhancing anti-tumor T cell immunity post-transplant, and in turn will help us design new generations of effective tumor vaccines in the setting of stem cell transplant.